The in vitro cell culture system has been widely used as a primary screening tool for evaluating anti-HIV-1 activity of microbicides. However, there is clearly a need to develop a vaginal and cervical tissue based in vitro system to test the cytotoxicity and antiviral activity in the context of the complete tissue matrix. We have recently developed a cervical tissue-derived organ culture model which mimics in vivo conditions and has been used to test microbicides for their ability to block HIV-1 transmission and measure inflammatory cytokines in response to microbicides and sexually transmitted infection-related bacteria. Our hypothesis is that a cervical tissue-based organ culture is an ideal system to test microbicides for toxicity in genital tissue and for its ability to block transmission of HIV-1 with varying phenotypic properties across the cervical epithelium in the presence of common environmental factors that are present in vagina, such as semen, vaginal fluid, and STI-related microorganisms. Specific aims of the project are: 1) Evaluation of -nucleoside reverse transcriptase inhibitor (NNRTI) 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) from Project 1 and the entry inhibitor antimicrobial peptide retrocyclin RC101 from Project 2 alone or in combination and their formulations for their ability to block HIV-1 transmission across the mucosa in a cervical tissue-based organ culture. 2) Assessment of cervical tissue inflammation and their changes in response to CSIC and RC101 from Projects 1 and 2, respectively, using the organ culture model. The expression of proinflammatory cytokines, such as 11-13, IL-6, IL-8 and TNF-a will be monitored by measuring their messages in the tissues by the real time PCR and secretion in the culture supernatant using the Luminex system; 3) Evaluation of anti-HIV activity of CSIC and RC101 under physiologically relevant conditions. CSIC and RC101 alone or in combination and their formulations will be evaluated for their antiviral activities in the presence of semen and vaginal fluid. In addition the organ culture model will be expanded to measure STIrelated microorganisms, such as Neisseria gonorrhoeae and Prevotella melaninogenica by measuring proinflammatory cytokine response. Microbicides will then be evaluated for their antiviral activity in the aresence of the proinflammatory cytokines induced by these microorganisms.The proposed studies in this Droject will complement various other projects in this U19 grant application by providing a valuable in vitro cervical tissue-based assay which will bridge between the microbicide development (Projects 1 and 2), monkey model (Project 4) and Formulation Core (Core B).